Abstract

The conditioning of cocaine's subjective actions with
environmental stimuli may be a critical factor in long-lasting relapse
risk associated with cocaine addiction. To study the significance of
learning factors in persistent addictive behavior as well as the
neurobiological basis of this phenomenon, rats were trained to
associate discriminative stimuli (SD) with the availability
of i.v. cocaine vs. nonrewarding saline solution, and then placed on
extinction conditions during which the i.v. solutions and
SDs were withheld. The effects of reexposure to the
SD on the recovery of responding at the previously
cocaine-paired lever and on Fos protein expression then were determined
in two groups. One group was tested immediately after extinction,
whereas rats in the second group were confined to their home cages for
an additional 4 months before testing. In both groups, the cocaine
SD, but not the non-reward SD, elicited strong
recovery of responding and increased Fos immunoreactivity in the
basolateral amygdala and medial prefrontal cortex (areas
Cg1/Cg3). The response reinstatement and Fos expression induced
by the cocaine SD were both reversed by selective
dopamine D1 receptor antagonists. The undiminished
efficacy of the cocaine SD to elicit drug-seeking behavior
after 4 months of abstinence parallels the long-lasting nature of
conditioned cue reactivity and cue-induced cocaine craving in
humans, and confirms a significant role of learning factors in the
long-lasting addictive potential of cocaine. Moreover, the results
implicate D1-dependent neural mechanisms within the
medial prefrontal cortex and basolateral amygdala as
substrates for cocaine-seeking behavior elicited by cocaine-predictive
environmental stimuli.

The conditioning of
cocaine's pharmacological actions with discrete environmental stimuli
has been implicated as a major factor in the abuse potential of this
drug (1). Both retrospective (2) and controlled laboratory studies
(3–5) show that such stimuli can evoke drug desire that may lead to
the resumption of drug use in abstinent individuals. Drug-related
stimuli may also elicit automatic responses that lead to drug-seeking
behavior and relapse without the intervention of distinct feelings of
craving (6, 7). Learned responses to drug-related stimuli, therefore,
represent a possibly critical element contributing to the chronic
relapsing nature of cocaine and other drug addiction (8, 9).

Consistent with a role of learning factors in the initiation of
drug-seeking behavior, cocaine-related stimuli can elicit strong
recovery of responding at a lever previously associated with i.v.
cocaine infusions in animal models of relapse (10, 11). However, little
information is available about the perseverance of the motivating
actions of such stimuli over prolonged periods of abstinence and the
neurobiological substrates mediating these effects. In humans, relapse
risk is typically greatest during the first 6 months of abstinence but
may persist for substantially longer periods of time (1, 8, 12). Better
understanding of the environmental conditions contributing to
long-lasting vulnerability to relapse and the neurobiological basis of
this phenomenon will be of substantial clinical benefit.

In work that has begun to address this issue, the efficacy of a
cocaine-predictive discriminative stimulus to elicit responding at a
previously active, cocaine-paired lever was found to remain unaltered
over 8 days of intermittent testing (11). These findings indicated that
the behavioral actions of cocaine-related environmental stimuli are
resistant to extinction despite repeated nonreinforced exposure to
these cues. Here, we have investigated the significance of drug-related
environmental stimuli in enduring vulnerability to relapse by examining
whether a drug-predictive stimulus retains its efficacy to induce
cocaine-seeking behavior after long-term abstinence. Additionally, to
identify sites that may participate in the control of conditioned
cocaine-seeking behavior, the rats' brains were examined for regions
showing neural activation after exposure to the cocaine cue as measured
by increased expression of Fos, the protein product of the
immediate-early gene c-fos (13, 14). Lastly, because of evidence that
cue-induced cocaine craving in humans is associated with neural
activation in dopamine-rich forebrain regions (4, 5, 15) and that
cocaine cues increase dopamine release within these regions in rats
(11), a third objective was to determine whether the behavioral effects
of the cocaine-predictive stimulus and its effects on Fos expression
are sensitive to pharmacological antagonism of dopamine
neurotransmission.

Materials and Methods

Subjects.

Male Wistar rats (Charles River Breeding Laboratories) weighing
250–300 g at the beginning of the experiment were used. Rats were
housed in groups of two or three in a temperature-controlled (22°C)
vivarium on a reverse 12-h light/12-h dark cycle with ad
libitum access to food and water. All procedures were conducted in
strict adherence to the National Institutes of Health guidelines.

Drugs.

Cocaine hydrochloride (National Institute on Drug Abuse,
Bethesda) was dissolved in sterile physiological saline at a
concentration of 0.25 mg/0.1 ml. Drug or vehicle solution was infused
at a volume of 0.1 ml over 4 s. SCH 39166 and SCH 23390
(Schering-Plough) were dissolved in ethanol (1
μg/μl) or methanol (1 μg/ml), respectively, diluted
with saline to a concentration of 30 μg/ml, and injected s.c.
25–30 min before test sessions.

Behavioral Training and Testing Procedures.

Surgical preparation for i.v. cocaine self-administration was
performed as described in ref. 16. Behavioral testing equipment and
i.v. self-administration protocols used were also identical to those in
ref. 16.

Self-Administration and Discrimination Training.

The self-administration and conditioning procedures have been
described in detail (11). Briefly, the purpose of these procedures was
to train rats to self-administer i.v. cocaine, while simultaneously
establishing discriminative stimuli (SD)
associated with cocaine availability vs. nonavailability. Once rats
developed stable levels of daily cocaine intake, they were subjected to
a discrimination learning regimen as follows. In three daily 1-h
sessions, either cocaine or saline was available as the only infusion
solution. Each training day included one saline and two cocaine
sessions conducted in random sequence. Sessions were initiated by
extension of the levers into the chambers and concurrent onset of the
respective SD which remained present until
termination of the session by retraction of the levers. The
SD associated with cocaine availability
(S+) consisted of an intermittent tone (7 kHz, 70
dB), whereas the SD, predictive of the saline
vehicle solution (that is, S− or nonreward)
consisted of continuous illumination of the self-administration
chamber's house light. To prevent accidental overdosing, drug
infusions were followed by a 20-s time-out period, signaled by
illumination of a white cue light during which time the lever remained
inactive. Saline infusions produced by lever-presses during
S− sessions were similarly followed by a 20-s
time-out period, signaled by a white noise (70 dB). In both the
S+ and S− conditions, two
levers were present. Only the right lever was active and, when
depressed, produced an i.v. infusion of the respective solution. The
left lever was inactive, but responses at this lever were recorded as a
measure of nonspecific activation. At 1 day after reaching a training
criterion, defined as stable cocaine-maintained responding (±10% over
3 successive days) and negligible responding during saline
availability, each animal was placed on extinction conditions.
Extinction sessions began by extension of the levers without
presentation of the SD. Responses at the
previously active lever activated the syringe pump motor only but had
no other programmed consequences. These sessions lasted 1 h and
were conducted once daily until a criterion of less than four responses
per session over 3 consecutive days was reached.

Cue Exposure Test Procedures.

After completion of extinction training, the rats were tested for the
degree of recovery of responding at the previously active lever induced
by exposure to either the cocaine S+ or
S− alone and the modification of the effects of
the cocaine S+ by SCH 39166 (10 μg/kg). These
tests were conducted under two conditions as follows. After reaching
the extinction criterion, each individual animal was randomly assigned
to either an immediate or delayed testing condition. Tests in rats
assigned to the immediate condition were conducted on the next day.
Rats designated for delayed testing remained in their home cages.
Before the delayed test (conducted 4 months after completion of the
first extinction phase), these animals were again briefly tested under
extinction conditions to reconfirm stable responding at or below the
extinction criterion. All tests were conducted by noncontingently
presenting rats with either the S+ or
S− throughout a single 1 h session without
the availability of cocaine or saline. Rats in both the immediate and
delayed test conditions were randomly divided into three groups for
testing with the S+, the
S−, or the S+ preceded by
the administration of SCH 39166. Presentation of the respective
SD began simultaneously with extension of the
levers and terminated with their retraction at the end of the test. As
during self-administration training, responses at the active lever were
followed by a 20-s signaled time-out period. After completion of the
tests, all rats were killed, and their brains were processed for Fos
immunohistochemistry as described below.

To confirm further an involvement of the D1
receptor in the behavioral effects of the cocaine
S+ and to establish the dose-dependency of
D1 antagonist treatments on cocaine-seeking
behavior, an additional group of rats was prepared for testing with
three doses of SCH 23390, a D1 antagonist analog
of SCH 39166. All experimental procedures were identical to those in
the immediate test condition described above, except that a
within-subjects design involving cue exposure tests at 3-day intervals
was used. All rats were tested with the S− and
subsequently with the S+ preceded by vehicle
administration before three drug tests involving exposure to the
S+ preceded by SCH 23390. To control for order
effects, rats received different doses of SCH 23390 (2.5, 5.0, and 10.0
μg/kg) in random sequence across the 3 drug-test days.

To examine whether the distribution of Fos immunoreactivity
associated with exposure to the cocaine S+
differs from that produced by cocaine itself, levels and distribution
of Fos were determined in rats after 60 min of i.v. self-administration
of cocaine. These animals were trained initially to respond for food
pellets on a fixed-ratio 1 schedule of reinforcement as described in
ref. 11. After acquisition of food-reinforced lever-pressing, the
animals were trained to self-administer cocaine on an fixed-ratio 1
schedule and received daily 2-h access to cocaine for a total of 2
weeks (cocaine chronic group). Because repeated cocaine exposure can
greatly desensitize cocaine-dependent Fos expression (17, 18), a 1-week
abstinence period was imposed before a final 60-min self-administration
session to allow for some recovery of cocaine-dependent Fos expression.
To provide a measure of acute cocaine effects, Fos immunoreactivity was
also measured in a group of previously drug-naive rats after the first
cocaine self-administration session (cocaine acute group) after
acquisition of food-reinforced responding. A third group was trained
and tested identically to the animals in the latter group but received
access to i.v. saline only. This group (saline group) served as a
control against which cocaine-induced changes in Fos immunoreactivity
were compared.

Immunohistochemistry.

Rats remained in the test chambers for 30 min after termination of the
cue exposure or cocaine self-administration sessions. The animals then
were deeply anesthetized by CO2 inhalation and
transcardially perfused with 20 ml of isotonic saline followed by a
solution consisting of 250 ml of 4% (vol/vol) paraformaldehyde in
0.1 M PBS (pH 7.4). Brains were removed from the skull, postfixed
overnight with 2% (vol/vol) paraformaldehyde in PBS at 4°C, rinsed
with PBS, and cryoprotected in 16% (vol/vol) sucrose/PBS solution
until sectioning. Brains were sliced coronally into 35-μm sections by
using a vibratome (Leica, Deerfield, IL) and stored at −20°C in 30%
(vol/vol) ethylene glycol/25% (vol/vol) glycerol/0.05 M
phosphate buffer. Sections were rinsed with PBS and incubated with a
Fos-specific rabbit polyclonal antibody (Santa Cruz Biotechnology) at a
dilution of 1:1,000 for 12 h at 4°C in PBS containing 1.5%
(vol/vol) goat serum and 0.3% Triton X-100. Sections then were
processed with an ABC kit (Vector Laboratories), and peroxidase
activity was revealed by 0.003%
H2O2 and 0.05%
3,3-diaminobenzidine-4 HCl and nickel enhanced, resulting in a
black-brown precipitate in specifically immunolabeled structures.
Sections were mounted on Superfrost+ slides (Fisher Scientific), air
dried, coverslipped, and analyzed by using a bright field microscope
(Axiophot, Zeiss). Images of the regions of interest were digitally
collected and counted by using a computer-assisted image analysis
system (mcid, Imaging Research, St. Catherines, ON,
Canada). For each of the sections, background was considered to be a
portion of the same area not expressing Fos-positive nuclei. For each
animal, the mean bilateral counts of Fos-immunoreactive nuclei for six
sections per brain region were determined. Brain regions were
identified by using the atlas of Paxinos and Watson (19).

Data Analysis.

Differences in the number of lever-press responses among rats
assigned to the S+ vs. S−
vs. S+ and SCH 39166/SCH 23390 cue exposure
conditions as well as differences between the number of cue-induced
responses vs. mean extinction responses at criterion (that is,
responses averaged across the final three sessions of the extinction
phase) were analyzed by mixed-factorial ANOVA. Differences among
individual means were verified subsequently by Newman–Keuls post hoc
tests and analysis of linear trends. Differences in the number of
Fos-positive neurons among groups assigned to the
S+, S−,
S+ & SCH 39166, and cocaine self-administration
conditions were analyzed separately for each brain region by one-way
ANOVA, followed by Newman–Keuls post hoc tests.

Results

Conditioning and Extinction Phase.

All rats (n = 34) developed stable cocaine
self-administration and ceased responding during saline sessions (Fig.
1a, left column). The mean
(±SEM) number of days to training criterion was (16.5 ± 1.17).
During subsequent sessions in which i.v. drug or vehicle infusions and
the corresponding SD were withheld, the rats
reached the extinction criterion (less than four responses per session
over 3 consecutive days) within 15.0 ± 2.8 days (Fig. 1a and b left and center columns).

Lever-press responses during the self-administration training,
extinction, and cue exposure test phases at the active
(a and b) and inactive (c,
d) lever. Self-administration (SA): cocaine-reinforced
(●) and saline/nonreinforced (○)
responses in the presence of discriminative stimuli respectively
associated with cocaine (S+) vs. saline (S−)
availability across the final 3 days of the self-administration phase
in rats assigned to the immediate (a and
c) and 4-month delayed (b and
d) cue exposure tests. Extinction (EXT): mean (±SEM)
number of responses during the last 3 days of the extinction phase.
Reinstatement: responses during exposure to the cocaine S+,
the S−, and the S+ preceded by administration
of SCH 39166 (S+SCH) during the immediate
(a and c) and 4-month delayed
(b and d) tests. In both test conditions,
presentation of the S+ increased the number of responses
over extinction performance, whereas the S− had no effect.
SCH 39166 significantly attenuated the effect of the S+,
and responding in this condition was not statistically different from
extinction levels. **, P < 0.01
(different from the extinction and S− conditions); ++,
P < 0.01) (different from the S+
condition).

Behavioral Effects of Reexposure to the Cocaine S+.

In rats assigned to the immediate test condition
(n = 18), presentation of the cocaine
S+ (n = 6) elicited a significant
increase in responding over extinction levels (P <
0.01) at the previously cocaine-paired lever, whereas the
S− (n = 6) had no effect (Fig.
1a, right column). The selective D1
antagonist SCH 39166 significantly attenuated the behavioral effect of
the S+ (P < 0.01), and the
number of responses in this condition (n = 6) was not
statistically different from extinction levels or responses in the
presence of the S− (post hoc analyses after
ANOVA: F2,15 = 29,79;
P < 0.001).

An identical pattern of effects was observed in rats
(n = 16) tested for the first time after 4 months of
abstinence (Fig. 1b). Presentation of the
S+ (in n = 6) but not the
S− (in n = 6) significantly
increased responding over extinction levels (P <
0.01). SCH 39166 inhibited the behavioral effect of the
S+ (P < 0.01), and the number of
responses in these animals (n = 4) did not differ
statistically from either extinction responses or responses in rats
presented with the S− (post hoc analyses after
ANOVA: F2,13 = 13.59;
P < 0.001). In both the immediate and delayed test
conditions, responding at an alternative lever that had been present
but inactive during the training phase was negligible (Fig. 1c and d).

In rats presented with the S+, the number of
Fos-immunoreactive nuclei was significantly increased within the
basolateral amygdala (immediate test: P < 0.05;
delayed test: P < 0.01) and medial prefrontal cortex
[prelimbic cortex (Cg3) and area I of the cingulate cortex (Cg1)]
(immediate test: P < 0.05; delayed test:
P < 0.05) compared with animals of the
S− control condition (Figs.
2 and 3).
No differences in Fos immunoreactivity between the groups exposed to
the cocaine S+ vs. S− were
found in any of the other targeted brain regions (Table
1).

Fos-immunoreactive nuclei in rats after exposure to the cocaine
S+, S−, and S+ preceded by
administration of SCH 39166 (S+SCH) in the
immediate (a and c) and 4-month delayed
(b and d) tests. Compared with the
S− control condition, the number of Fos-immunoreactive
nuclei was significantly increased after exposure to the cocaine
S+ in the basolateral amygdala and medial prefrontal cortex
(see also caption of Fig. 3), both in the immediate and delayed testing
conditions. SCH 39166 reversed the effects of the cocaine
S+ on Fos expression. *, P <
0.05; **, P < 0.01 (different from
the S− condition). †, P < 0.05, ‡,
P < 0.01 (different from the S+
condition).

Dose Dependency of D1 Antagonist Effects on
S+-Induced Responding.

In rats (n = 5) pretreated with the
D1 antagonist SCH 23390, the recovery of
responding induced by the cocaine S+ was
attenuated (Fig. 4). Responses after SCH
23390 treatments differed significantly from those produced by vehicle
treatment (P < 0.05) and from cocaine-reinforced
responses (P < 0.05) during the self-administration
phase (Newman–Keuls post hoc tests after ANOVA:
F7,28 = 9.33; P <
0.0001). The dose-dependency of the drug treatment effects was
confirmed by analysis of the linear trend across the five means of the
dose-response function (0–20 μg/kg). This analysis revealed that
the suppression of response by SCH 23390 increased significantly with
increasing doses (F1,4 = 17.15;
P < 0.05).

Effects of the D1 antagonist SCH 23390 on response induced
by the discriminative stimulus for cocaine (S+). SCH 23390
dose-dependently reversed the effects of the S+. For
comparison, the figure also shows the average number of responses
during the last 3 days of the self-administration (SA) and extinction
(EXT) phases, as well as responses in the presence of the stimulus
associated with nonreward (S−). *,
P < 0.05, significant linear trend over dose
levels; +, P < 0.05, different from the EXT and
S− conditions; &, P < 0.01, different
from cocaine-reinforced responses (SA).

Cocaine-Dependent Fos Expression in Self-Administering Rats.

The mean (±SEM) number of cocaine infusions during the 1-h
self-administration session before rats were killed was 8.83 (±1.17)
in rats given access to cocaine for the first time (cocaine acute
group; n = 6), and 15.4 (±1.89) in cocaine-experienced
rats (cocaine chronic group; n = 5). Rats assigned to
the control condition (saline group; n = 6) received an
average of 6.5 (±2.89) vehicle infusions. Cocaine self-administration
significantly increased the number of Fos-positive nuclei over levels
in saline control rats (see Table 1, self-administration). In
previously drug-naive animals, Fos immunoreactivity was increased
ubiquitously throughout dopamine-rich brain regions including the
dorsal striatum, shell and core regions of the nucleus accumbens, the
central nucleus of the amygdala and medial prefrontal cortex, as well
as within thalamic and hypothalamic nuclei. In rats previously trained
to self-administer cocaine, increases in Fos immunoreactivity were
confined to the core of the nucleus accumbens, dorsolateral striatum,
and agranular cortex.

Discussion

The results confirm that discriminative stimuli associated with
the availability and self-administration of cocaine can elicit strong
drug-seeking behavior in rats as measured by the recovery of
extinguished responding at a previously cocaine-paired lever. Of
particular significance is the finding that the cocaine
S+ was effective in reinstating drug-seeking
behavior not only in rats tested immediately after the initial
extinction phase, but that the behavioral effects of this cue were
undiminished in rats subjected to an additional 4 months of abstinence.
This finding documents that the motivating actions of environmental
cues that act as discriminative stimuli for drug availability can
remain intact over a substantial length of time and, therefore, that
such stimuli may play a significant role in long-lasting relapse risk.

Exposure to the cocaine S+ selectively
increased the number of Fos-immunoreactive neurons in the basolateral
amygdala and medial prefrontal cortex. Fos immunoreactivity in the
striatum and nucleus accumbens, brain regions implicated in the
psychomotor stimulant effects of cocaine, was not altered by the
cocaine cue. There were also no differences in Fos expression in the
motor cortex between the groups exposed to the cocaine
S+ vs. S−. Thus, the
increased neural activation by the S+ within the
basolateral amygdala and medial prefrontal cortex is unlikely to be
secondary to motoric activation associated with increased
lever-pressing in the animals exposed to the S+.
This conclusion is supported also by a related finding showing that Fos
protein expression in the basolateral amygdala and cingulate cortex is
increased after exposure to a cocaine-paired environment in rats that
are not given the opportunity to emit lever-press responses (20).

The undiminished efficacy of the cocaine cue to elicit
cocaine-seeking behavior in the delayed test parallels the often
long-lasting nature of conditioned cue reactivity and cue-induced
cocaine craving in humans (for an example, see ref. 21). An important
observation in this regard is that distribution of Fos expression
induced by the cocaine S+ also closely parallels
data from PET and fMRI brain-imaging studies in humans showing that
cue-induced cocaine craving is associated with selective neural
activation of the amygdala and cingulate cortex (4, 5, 15). The
consistency between neuroanatomical sites showing activation during
cocaine craving in brain-imaging studies and the distribution of Fos
immunoreactivity associated with exposure to the cocaine
S+ in the present study suggests that these
procedures may provide an effective research tool for the elucidation
of the neural mechanisms underlying cocaine craving and long-lasting
relapse risk.

The selective dopamine D1 antagonist SCH
39166 concurrently reversed the increases in Fos immunoreactivity and
cocaine-seeking behavior induced by the cocaine
S+ in both the immediate and delayed test
condition. In addition, the behavioral effects of the cocaine cue were
dose-dependently blocked by a second D1
antagonist, SCH 23390. The attenuation of cue-induced responding by SCH
39166 and SCH 23390 cannot readily be attributed to nonspecific
impairments in motor performance. Low-dose D1
antagonist treatments decrease the direct reinforcing actions of
cocaine without interfering with motoric behavior (22). More
importantly, with respect to a role of the D1
receptor in conditioned cocaine-seeking behavior, SCH 23390
dose-dependently reduced responding induced by a light cue conditioned
to cocaine but left intact behavior maintained by a conditioned
stimulus associated with a food reinforcer (23). Interestingly, SCH
39166 (ecopipam) has been shown to attenuate the euphoric effects of
cocaine selectively in humans (that is, without inducing sedation or
nonspecific anhedonic effects) and, therefore, may have therapeutic
potential as a cocaine antagonist (24). Considering the present
results, the D1 receptor may be a promising
target not only for treatment approaches aimed at antagonizing the
euphoric effects of cocaine, but also for the blunting of cocaine
craving and prevention of relapse.

The increase in Fos expression within the basolateral amygdala
associated with exposure to the cocaine S+ is
consistent with evidence that this brain region is critical for the
capacity of stimuli conditioned to both drug and nondrug reinforcers to
affect behavior. Lesions of the basolateral amygdala reduce
cocaine-seeking behavior elicited (25, 26) or maintained (27) by
cocaine-related stimuli, prevent conditioned opiate withdrawal
responses (28), and impair conditioned behavior maintained by stimuli
associated with sexual reward (29). Recent findings suggest that the
conditioned behavioral effects of cocaine-related stimuli may depend on
activation of dopamine neurotransmission within the basolateral
amygdala (11). The concurrent reversal of
S+-induced cocaine-seeking behavior and neural
activation by D1 receptor antagonists supports
this possibility and, in particular, implicates
D1-dependent neural mechanisms within the
basolateral amygdala in the control of drug-seeking behavior by
external stimuli. The immunohistochemical findings are consistent also
with the current understanding of the role of the medial prefrontal
cortex in associative learning. Medial prefrontal cortical areas,
including the regions sampled here, have been implicated in attentional
performance, discrimination learning, and the learning of stimulus
attributes predicting reward or no reward (30–33), functions that can
be presumed to be essential for the mediation of associations between
the subjective effects of cocaine and environmental stimuli. Indeed,
contextual stimuli associated with cocaine reward consistently have
been found to produce elevations in Fos protein expression within the
cingulate cortex in other experimental paradigms (20, 34). The present
results further confirm these findings but, in addition, provide direct
evidence for a role of medial prefrontal cortical regions in mediating
the reinstatement of drug-seeking behavior by drug-associated
environmental stimuli. Moreover, as in the case of the basolateral
amygdala, the results suggest that these actions may involve
D1-dependent neural mechanisms.

A secondary objective of this study was to determine whether the
pattern of neural activation associated with drug-seeking behavior
induced by the cocaine S+ differs from that
produced by the unconditioned effects of cocaine in self-administering
rats. In the basolateral amygdala, Fos immunoreactivity was increased
selectively after exposure to the cocaine S+ and
was not altered either in rats self-administering cocaine acutely or in
rats with a history of cocaine self-administration. In contrast,
increases in Fos expression in the medial prefrontal cortex were
produced by the cocaine S+ as well as by cocaine.
Cocaine-induced neural activation in this brain region was, however,
seen only in previously drug-naive rats that showed widespread
increases in Fos immunoreactivity. No overlap was found between the
effects of cocaine and the cocaine S+ in rats
with a history of cocaine self-administration. Neural activation in the
latter group was restricted to the core of the nucleus accumbens,
dorsal striatum, and agranular cortex, suggesting that these brain
regions may perhaps recover more rapidly from the desensitization of
cocaine-dependent Fos expression associated with repeated cocaine
treatments (17, 18) than other sites that showed elevated Fos
expression in rats self-administering cocaine acutely. However, the
differences between the acute and chronic cocaine groups are not
readily accounted for by a differential recovery from desensitization,
because factors such as arousal, novelty, and stress associated with
i.v. cocaine infusions can be presumed to have contributed to the
pattern of neural activation in cocaine-naive rats. Indeed, brain
regions activated by cocaine in the chronic rather than the acute group
are more likely to represent sites that are more specifically involved
in the psychostimulant and rewarding effects of the drug. In agreement
with this presumption, it has been shown that cocaine does not increase
the expression of Fos-related antigens that are known to be induced
selectively by chronic, but not acute, cocaine exposure (18) within the
basolateral amygdala and cingulate cortex of rats with a history of
chronic self-administration (35). Thus, whereas the cingulate and
prelimbic cortex show activation by cocaine in drug-naive rats, these
brain regions do not seem to be recruited for the mediation of the
primary reinforcing actions of cocaine in animals trained to
self-administer the drug. This interpretation is not at variance with a
report that Fos protein levels in the cingulate cortex are elevated
both after reexposure to a drug-paired environment and by cocaine
priming injections in previously cocaine-self-administering rats (20).
Priming injections typically signal the response-contingent
availability of a drug. Under these conditions, cocaine acts as a
discriminative stimulus, and this action is likely to be similar to
that of drug-related stimuli or environments but different from the
reinforcing effects of the drug in actively self-administering animals.
It must, nonetheless, be emphasized that the failure to observe
increases in Fos immunoreactivity in rats self-administering cocaine
does not necessarily rule out an involvement of the basolateral
amygdala and medial prefrontal cortex in the primary reinforcing
effects of cocaine. However, the selective neural activation of these
brain regions by the cocaine S+ is consistent
with evidence that lesions of the basolateral amygdala or medial
prefrontal cortex interfere with many of the conditioned behavioral
effects of cocaine-associated stimuli without altering behavior
reinforced by the drug itself (25–27, 36). When interpreted in the
context of this literature, the immunohistochemical data provide
further support for a specific role of the basolateral amygdala and
medial prefrontal cortex in the mediation of learned responses to
cocaine-associated stimuli and strengthen the hypothesis that the
conditioned behavioral effects of cocaine-associated contextual stimuli
recruit different neuroanatomical substrates than the primary
reinforcing actions of cocaine (4, 20, 27).

In conclusion, the results confirm that the efficacy of
cocaine-predictive discriminative stimuli to elicit drug-seeking
behavior remains intact over prolonged periods of abstinence and, by
extension, support the hypothesis that learned responses to
drug-related stimuli are an important factor in long-lasting
vulnerability to relapse. Moreover, the data implicate the medial
prefrontal cortex and basolateral amygdala as candidate sites for the
mediation of cue-induced cocaine-seeking behavior and suggest that the
D1 receptor may represent an important
neuropharmacological substrate for the motivating effects of
cocaine-related stimuli.

Acknowledgments

We thank Cindy Simpson for technical assistance with Fos
immunohistochemistry, Daniele Vitali for valuable support with image
analysis, and Mike Arends for assistance with the preparation of the
manuscript. This work was supported by National Institute on Drug Abuse
Grants DA 08467 and DA 07348 (to F.W.). This article is publication
number 13052-NP from The Scripps Research Institute.

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